The synergistic mechanism of fibroblast growth factor 18 and integrin β1 in rat abdominal aortic aneurysm repair

Abstract

Background: Abdominal aortic aneurysms have a high mortality rate. While surgery is the preferred treatment method, the biological repair of abdominal aortic aneurysms is being increasingly studied. We performed cellular and animal experiments to investigate the simultaneous function and mechanism of fibroblast growth factor 18 and integrin β1 in the biological repair of abdominal aortic aneurysms.

Methods: Endothelial and smooth muscle cells of rat arteries were used for the cellular experiments. Intracellular integrin β1 expression was regulated through lentiviral transfection. Interventions with fibroblast growth factor 18 were determined according to the experimental protocol. Several methods were used to detect the expression of elastic fiber component proteins, cell proliferation, and migratory activity of endothelial and smooth muscle cells after different treatments. For animal experiments, abdominal aortic aneurysms were induced in rats by wrapping the abdominal aortae in sterile cotton balls soaked with CaCl₂ solution. Fibroblast growth factor 18 was administered through tail vein injections. The local expression of integrin β1 was regulated through lentiviral injections into the adventitia of the abdominal aortic aneurysms. The abdominal aortae were harvested for pathological examinations and tensile mechanical tests.

Results: The expression of integrin β1 in endothelial and smooth muscle cells could be regulated effectively through lentiviral transfection. Animal and cellular experiments showed that fibroblast growth factor 18 + integrin β1 could improve the expression of elastic fiber component proteins and enhance the migratory and proliferative activities of smooth muscle and endothelial cells. Moreover, animal experiments showed that fibroblast growth factor 18 + integrin β1 could enhance the aortic integrity to withstand stretch of aortic aneurysm tissue.

Conclusion: Fibroblast growth factor 18 + integrin β1 improved the biological repair of abdominal aortic aneurysms in rats by increasing the expression of elastic proteins, improving the migratory and proliferative abilities of endothelial and smooth muscle cells, and improving aortic remodeling.

Keywords: Abdominal aortic aneurysm; Biological repair; Endothelial cells; Fibroblast growth factor 18; Integrin β1; Smooth muscle cells.

Fig. 1

a FGF18 promotes the expression of elastic fiber component proteins in ECs in a concentration-dependent manner; after 24 h of treatment with FGF18 solution of different concentrations, the expression of elastin, fibulin-5, and fibrillin-1 in ECs increased with the increasing FGF18 solution concentration; **p < 0.01 versus the 0 ng/mL group. ***p < 0.005 versus the 0 ng/mL group; b FGF18 promotes the expression of elastic fiber component proteins in ECs in a time-dependent manner; when the concentration of the FGF18 solution was fixed at 100 ng/mL, the expression of the elastic fiber component proteins in ECs increased with the increase in treatment duration; **p < 0.01 versus the 0 h group. ***p < 0.05 versus the 0 h group; c Effects of FGF18 + Itgβ1 on elastic fiber component proteins and Itgβ1 expression in Ecs; the expression of elastic fiber component proteins increased in the groups treated with FGF18. After treatment with FGF18, ECs in the NC + LV-Itgβ1 group showed a higher expression of elastic fiber component proteins than the other groups; FGF18 did not affect the expression of Itgβ1; **p < 0.01 versus the control groups (groups that were not treated with FGF18)

Fig. 2

Effects of FGF18 on elastin expression in SMCs. a There was no significant difference in elastin expression between the SMCs treated with 100 ng/mL of FGF18 solution and the SMCs that were not treated with the FGF18 solution (p = 0.163); there was no significant difference in elastin expression between the SMCs treated with 200 ng/mL of FGF18 solution and the SMCs treated with 400 ng/mL of FGF18 solution (p = 0.379). b There was no significant difference in the expression of elastin at different time points after treatment with 100 ng/mL FGF18 solution (p > 0.05). c FGF18 + Itgβ1 can promote the expression of elastin; in the Con group (not treated with FGF18), there was no significant difference in elastin expression amongst the NC, NC + LV-con, NC + LV-Itgβ1-RNAi, and NC + LV-Itgβ1 groups (p > 0.05); there was also no significant difference in elastin expression amongst the NC, NC + LV-Con, and NC + LV-Itgβ1-RNAi groups after FGF18 treatment (p > 0.05); after treatment with FGF18 (100 ng/mL, 24 h), the elastin expression in SMCs in the NC + LV-Itgβ1 group was higher than that in the other three groups (p < 0.05). ***p < 0.001 versus the NC group (treated with FGF18); (d) FGF18 + Itgβ1 promoted the expression of elastic fiber component proteins in SMCs; the expression levels of elastin, fibulin-5, and fibrillin-1 in SMCs in the NC + LV-Itgβ1 group showed a positive relationship with the concentration of the FGF18 solution; The WB gel of a, b, c, and d were cut prior to hybridization with antibodies; **p < 0.05 versus the 0 ng/mL group. ***p < 0.001 versus the 0 ng/mL group

Fig. 3

CCK-8 assay for detecting the proliferative activity of ECs and SMCs. a Ecs: the proliferative activity of ECs was significantly enhanced after treatment with FGF18 (p < 0.05); the proliferative activity of ECs was higher in the NC + LV-Itgβ1 + FGF18 group than in the NC + LV-Itgβ1, NC + FGF18, and NC groups (p < 0.05); amongst the groups that were not treated with FGF18, the proliferative activity of ECs in the NC + LV-Itgβ1 group was the strongest, whereas that in the NC + LV-Itgβ1-RNAi group was the weakest. b SMCs: FGF18 alone could not improve the proliferative activity of SMCs in the NC and NC + LV-Con groups (p = 0.127, 0.082); FGF18 improved the proliferative activity of SMCs in the NC + LV-Itgβ1 group (p = 0.032); the proliferative activity of SMCs was higher in the NC + LV-Itgβ1 group than in the NC and NC + LV-Con groups (p = 0.012, 0.024); the proliferative activity of SMCs was higher in the NC + LV-Itgβ1 + FGF18 group than in the NC, NC + LV-Itgβ1, and NC + FGF18 groups (p < 0.05)

Fig. 4

a Survival curves of rats in different groups; mortality of the Sham group was 0 within 4 weeks after surgery; three rats in the experimental groups died within 2 weeks after surgery, and the overall mortality of the experimental groups within 4 weeks after surgery was 6.25% (3/48). b The AAA incidence rate of rats in different groups: forty rats developed AAA: 10 in the AAA group, 10 in the AAA + FGF18 group, 11 in the AAA + LV-Itgβ1 group, and nine in the AAA + FGF18 + LV-Itgβ1 group; there was no significant difference in the morbidity and mortality between the experimental groups (p > 0.05). (c, d) The abdominal aorta dilation rate of different groups at 0–4 weeks after the operation. There was no significant difference in the abdominal aorta dilation rate between groups 2 weeks after the operation (p > 0.05); four weeks after the operation, the abdominal aorta dilation rate of the AAA + FGF18 + LV-Itgβ1 group was the lowest, whereas that of the AAA group was the highest; there was a significant difference between them (p < 0.01); **p < 0.01

Fig. 5

a Immunohistochemical staining: Itgβ1 was mainly expressed in the intima and media of the aortic wall; Itgβ1 was strongly expressed (+++) in the Sham group; Itgβ1 expression in the AAA + FGF18 and AAA groups was similar; both were weakly positive (+); compared with the AAA group, Itgβ1 expression was significantly higher in the AAA + LV-Itgβ1 and AAA + FGF18 + LV-Itgβ1 groups. Both groups were moderately positive (++). b HE staining: the aortic wall of the Sham group was normal; the structure of the aortic wall in the AAA group was destroyed, and rupturing of the elastic fibers and sparse arrangement were observed; the elastic fibers of the AAA + FGF18 group were not damaged to some extent; the aortic media in the AAA + LV-Itgβ1 + FGF18 group was thinner than that in the Sham group, and the overall structure of the aortic wall was relatively intact. c EVG staining: The structure of the abdominal aortic wall in the Sham group was normal, and the elastic fibers were continuous and regularly corrugated; the elastic fibers of the AAA group were broken and irregularly arranged; AAA + FGF18 group: the elastic fibers increased, and a sparse arrangement and local fractures were osberved; the white tip represents the irregularly arranged elastic fibers; the elastic fibers of the AAA + LV-Itgβ1 group were dense and regularly arranged, but local fractures were observed; the elastic fibers of the AAA + FGF18 + LV-Itgβ1 group were continuous and densely arranged; bar = 500 μm, 100 μm

Fig. 6

Tensile mechanical test of rat AAA tissues. Sham group: the aortic tensile resistance was the strongest, and the average maximum tensile force was 970 ± 26 mN; AAA group: the aortic tensile resistance was the lowest, and the average maximum tensile force was 111 ± 25 mN; the mean aortic maximum tensile forces of the AAA + LV-Itgβ1 and AAA + FGF18 groups were 254 ± 21 mN and 266 ± 23 mN, respectively; the aortic integrity to withstand stretch of the AAA + FGF18 + LV-Itgβ1 group was significantly higher than that of the AAA + LV-Itgβ1 and the AAA + FGF18 groups (p = 0.041, 0.032), and the average maximum stretching force was 493 ± 29 mN